Reactivity of glycoconjugate in membrane system II: Can a neutral glycolipid function as lectin receptor in the presence of gangliosides in plasma membrane?

To examine how surface Potential controls the reactivity of glycoconjugates at cell surface, the interaction of galactose-sPecific lectinse.g. peanut agglutinin,Ricinus cummunis agglutinin with liPosomes bearing asialo GM₁ were studied in the Presence of varying amount of ganglioside mixture, GM_n. The Presence of 5% GM_n causes comPlete slowing down of PreciPitin reaction and thereby make carbohydrate moiety of asialo GM₁ comPletely inaccessiblei.e. ‘cryPtic’. In contrast the Presence of 1–2% GM_n enhances the aPParent rate and amPlitude of the PreciPitin reaction as surface Potential becomes more negative. The relevance of the findings has been discussed in relation to the exPression and involvement of the cell-surface sialic acid residues during develoPment and differentiation.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Preparative separation of small molecular weight peptides from casein hydrolysate using gel filtration and immobilized metal ion affinity chromatography

A two step method consisting of a gel filtration step, followed by a Immobilized Metal Affinity Chromatography (IMAC) step using a IDA-Cu coupled Sephadex G-25 column, on a preparative scale is described for the group separation of peptides from a casein hydrolysate. The 48 groups of peptides thus separated are further characterised by RP-HPLC and amino acid analysis. Some peptides after the analytical RP-HPLC step are further characterised by sequencing. An insight into the mechanism of retention on IMAC of the peptides is attempted. In such complex mixtures as casein hydrolysate, the peptide-peptide interaction can mask the potential sites of interactions in a single peptide. The results obtained using volatile buffers as eluents show the possibility of using IMAC step as an alternative to obtain gram quantities of group of peptides free of salts from complex protein hydrolysates.     

Noncytotoxic Dy3+-activated glass emits cool white light for near ultraviolet-based white light-emitting diodes,visible lasers,and biomedical applications

Using the melt quenching technique, a lithium zinc borate glass (LZB) system with trivalent dysprosium ions (Dy³⁺) was synthesized, and the luminescence and lasing properties of these materials were examined for the generation of white light. Structural investigation through X-ray diffraction revealed that the prepared glass had an amorphous nature. The optimized glass containing 0.5 Dy³⁺ had a direct optical band gap of 2.782 eV and an indirect optical band gap of 3.110 eV. A strong excitation band at 386 nm (⁶H_15/2→⁴I_13/2) was recognized in the ultraviolet (UV) light region of its excitation spectrum. Emission bands could be seen in the photoluminescence spectrum at 659, 573, and 480 nm under the 386 nm excitation. These transitions of emission resembled electronic transitions such as (⁴F_9/2→⁶H_11/2), (⁴F_9/2→⁶H_13/2), and (⁴F_9/2→⁶H_15/2). In a pristine glass matrix, the higher intensity ratio of yellow to blue can result in the production of white light. The optimized Dy³⁺ ion concentration was observed to be 0.5 mol%. In addition, an analysis of lifetime decay was conducted for all synthesized glasses, and their decay trends were systematically investigated. Noticeably, we assessed the photometric parameters and found that they were close to the white light standard. Furthermore, a cytotoxicity study was carried out using lung fibroblast (WI-38) cell lines for the optimized 0.5Dy³⁺-doped LZB glass and it appeared to be noncytotoxic. It is clear from the results that the noncytotoxic LZB glass doped with 0.5 Dy³⁺ ions could be a suggestive choice for the manufacture of white light-emitting diodes and lasers using near-UVs.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.     

L1210/B23.1 cells express equilibrative, inhibitor-sensitive nucleoside transport activity and lack two parental nucleoside transport activities.

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L1210/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydorxy-3-nonyl)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleotide transporters). The selection medium did not allow es activity and selected against cells that expressed the Na(+)-linked cif process. Cells of the L1210/B23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of [3H]formycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L1210/B23.1 cells indicates that (i) the mutation/selection procedure impaired or deleted the Na(+)-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.     

Molecular docking analysis of beta-catenin with compounds derived from Lycopersicon esculentum

Beta-catenin is linked with colorectal cancer (CRC). Therefore, it is of interest to design and develop novel compounds to combat CRC. Hence, we document compounds (chlorogenic acid, gallic acid, protocatechuic acid, quercetin and vanillic acid) from Lycopersicon esculentum with optimal binding features for further consideration.